Compound of glycosaminoglycan and its preparation method as well as application

ABSTRACT

The present invention is related to a compound conjugating a drug with a glycosaminoglycan, such as hyaluronic acid (HA), where the drug is useful for the treatment of diseases such as inflammation, auto-immune disease, allergy, infection and preferably cancer. The conjugated compound of the present invention can increase the concentration of drug at the specific site of disease by an interaction of the glycosaminoglycan used as target drug delivery carrier and the CD44 cell surface receptor, then enhancing the therapeutic efficacy and reducing the systemic side effect of the site-delivered drug.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/871,352, filed on Aug. 29, 2013, the content of which ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a compound consisting of aglycosaminoglycan conjugate with a drug and to a preparation thereof

2. Description of the Prior Arts

The extracellular matrix (ECM) is a dynamic assemblage of interactingmolecules that regulate cell functions and interactions in response tostimulation. One class of extracellular matrix macromolecules, theglycosaminoglycans, are molecules known to be involved in a wide arrayof both normal and abnormal biological processes, including cellmigration, differentiation, proliferation, immune response andcytoskeletal organization.

Glycosaminoglycans (GAGs) are unbranched chains composed of repeatingdisaccharide units. These disaccharide units always contain an aminosugar (N-acetylglucosamine or N-acetylgalactosamine), which in mostcases is sulfated, with the second sugar usually being an uronic acid(glucuronic or iduronic). GAGs are highly negatively charged because ofthe presence of carboxyl or sulfate groups on most of their sugarresidues. As such they are strongly hydrophilic. GAGs tend to adopthighly extended conformations and form matrices that are space fillingand resistant to compressive forces. Four main groups of GAGs have beendistinguished by their sugar residues, the type of linkage between theseresidues, and the number and location of sulfate groups. They include:(1) hyaluronan, (2) chondroitin sulphate and dermatan sulfate, (3)heparan sulfate and heparin, and (4) keratan sulfate.

Hyaluronan (also called hyaluronic acid or hyaluronate or HA) is thesimplest of GAGs. It consists of a regular repeating sequence ofnon-sulfated disaccharide units, specifically N-acetylglucosamine andglucuronic acid. Its molecular weight can range from 400 daltons (thedisaccharide) to over millions of daltons. It is found in variableamounts in all tissues, such as the skin, cartilage, and eye, and inmost, if not all, fluids in adult animals. It is especially abundant inearly embryos. In articular cartilage, HA can form a large aggregatewhich is important for the function of cartilage. Furthermore, cellmotility and immune cell adhesion is mediated by the cell surfacereceptor RHAMM (Receptor for Hyaluronan-Mediated Motility) and CD44.

HA is synthesized directly at the inner membrane of the cell surfacewith the growing polymer extruded through the membrane to the outside ofthe cell as it is being synthesized. Synthesis is mediated by a singleprotein enzyme, hyaluronan synthetase (HAS). By contrast, other GAGs aresynthesized inside the cell in the Golgi apparatus, possibly inassociation with some core protein, and then released by exocytosis. HAdegradation in vertebrate tissues in vivo is mediated by hyaluronidase,and exoglycosidases that remove sugars sequentially. Mammalian-typehyaluronidases have both hydrolytic and transglycosidase activities andcan degrade HA and chondroitin. In connective tissue, the water ofhydration associated with HA creates spaces between tissues, thuscreating an environment conducive to cell movement and proliferation. HAplays a key role in biological phenomena associated with cell motilityincluding rapid development, regeneration, repair, embryogenesis,embryological development, wound healing, angiogenesis, andtumorigenesis.

CD44 (also known as Pgp-1, Hermes-3, HCAM, ECMR III) is a widelyexpressed glycoprotein with a molecular weight of 85 to 90 kDa. CD44 isa major cell surface receptor for the glycosaminoglycan, hyaluronic acid(HA). CD44 binds HA specifically, although certain chondroitin-sulfatecontaining proteoglycans may also be recognized. CD44 plays a role invarious cellular and physiological functions, including adhesion to andmigration on HA, HA degradation and tumor metastasis. CD44 has also beenshown to play a role in extracellular matrix binding, cell migration,lymphocyte activation, lymphocyte homing, and proliferation of bronchialsmooth muscle cell (Gunthert et al., 1991, A new variant of glycoproteinCD44 confers metastatic potential to rat carcinoma cells, 5;65(1):13-24). The CD44 receptor shows a complex pattern of alternativesplicing in its variable region of the extracellular domain. CD44appears to be a particularly important leukocyte receptor for HA and maytherefore have a role in the pathogenesis of asthma. In addition, levelsof HA, which were increased during experimental asthma in control micewere markedly attenuated in the antibody-treated mice, supporting a rolefor CD44 in HA metabolism (specifically in the breakdown of highmolecular weight HA to pro-inflammatory low molecular weight forms).This may be particularly important because HA-derived oligosaccharidescan bind and activate Toll-like receptor. Clearly, the most impressiveaspect of the results is the profound magnitude of the beneficialeffects of anti-CD44 treatment.

HA-CD44 interactions may play an important role in development,inflammation, T cell recruitment and activation, lung inflammation, andtumor growth and metastasis.

Mice with a targeted deletion of standard CD44 and all isoforms developnormally. Studies have suggested an important role for CD44 ininflammatory states such as rheumatoid arthritis and the extravasationof T cells to sites of tissue inflammation. CD44 may have an importantrole in the recruitment of inflammatory cells in allergen-induced airwayinflammation in mice. CD44 has been suggested to play a critical role inregulating chronic inflammation, suggesting that CD44 may have acritical role in regulating macrophage activation independently ofinteractions with HA (Dianhua Jiang, Hyaluronan in Tissue Injury andRepair, Annu. Rev. Cell Dev. Biol. 2007; 23:435-61).

The ability to control inflammatory and immune responses is central tothe therapy of a wide spectrum of diseases. CD44 supports the adhesionof activated lymphocytes to endothelium and smooth muscle cells.Furthermore, ligation of CD44 induces activation of both inflammatoryand vascular cells. HA, the principal ligand for CD44, is upregulated inatherosclerotic lesions of apoE-deficient mice and thelow-molecular-weight proinflammatory forms of HA stimulate VCAM-1(vascular cell adhesion molecule-1) expression and proliferation ofcultured primary aortic smooth muscle cells, whereashigh-molecular-weight forms of HA inhibit smooth muscle cellproliferation. Gal-9 (Galectin-9) can reduce AHR (airwayhyperresponsiveness) as well as Th2-associated airway inflammation.Furthermore, administration of Gal-9 as well as anti-CD44 monoclonalantibody inhibited the infiltration of peripheral blood Th2 cells intothe airway. Interestingly, Gal-9 directly bound the CD44 adhesionmolecule and inhibited interactions with HA. Consistent with the conceptthat CD44-HA interactions mediate the migration of T cells into thelung, Gal-9 blocked CD44-dependent adhesion of BW5147 mouse T cells toHA. It was concluded that Gal-9 inhibits allergic inflammation of theairway and AHR by modulating CD44-dependent leukocyte recognition of theextracellular matrix (Shigeki Katoh, et al., Galectin-9 InhibitsCD44-Hyaluronan Interaction and Suppresses a Murine Model of AllergicAsthma, American Journal of Respiratory and Critical Care Medicine, 2007Jul. 1; 176(1):27-35).

The CD44 will be expression at the autoimmune disease likes systemiclupus erythematosus (SLE), Rheumatoid arthritis, Sjögren's syndrome,inflammatory bowel disease (IBD), Ankylosing spondylitis, Psoriaticarthritis, Psoriasis, Dermatomyosistis, Vasculitis, and Behcet'sdisease. Systemic lupus erythematosus is a prototype autoimmune diseasethat affects multiorgan systems. Accumulating evidence suggests thathyaluronan and its interaction with its cell surface receptor CD44 playsan important role in mediating pathogenic mechanisms in SLE (Yung S andChan T M, 2012, The Role of Hyaluronan and CD44 in the Pathogenesis ofLupus Nephritis, Autoimmune Dis. Volume 2012 (2012), Article ID 207190,9 pages). Rheumatoid Arthritis (RA) is a common autoimmune disorder thatresults in inflammation of the synovial joints of patients. Though RAaffects approximately 1% of the population and is classified as anautoimmune disorder, the molecular event(s) which initiate the evasionof tolerance remain speculative and unconfirmed (Patrick J. Mott, CD44Antibodies and Immune Thrombocytopenia in the Amelioration of MurineInflammatory Arthritis, PLoS One, 2013, 8(6): e65805). While Sjögren'ssyndrome (SS) is more common than related autoimmune disorders, such assystemic lupus erythematosus (SLE) and rheumatoid arthritis (RA),scientific and medical research in SS has lagged behind significantly.This is especially true in the field of SS genetics, where efforts todate have relied heavily on candidate gene approaches (John A. Ice,Genetics of Sjögren's syndrome in the genome-wide association era, JAutoimmun. 2012 August; 39(1-2):57-63). Ankylosing Spondylitis (AS) is acommon inflammatory rheumatic disease with a predilection for the axialskeleton, affecting 0.2% of the population. Current diagnostic criteriarely on a composite of clinical and radiological changes, with a meantime to diagnosis of 5 to 10 years (Roman Fischer, 2011, Discovery ofCandidate Serum Proteomic and Metabolomic Biomarkers in AnkylosingSpondylitis, Mol Cell Proteomics, 2012 February; 11(2):M111.013904). Theresults suggest that circulating T lymphocytes bearing activated CD44are elevated under conditions of chronic inflammation and that these mayrepresent a pathogenically important subpopulation of activatedcirculating cells that may provide a reliable marker for autoimmune orchronic inflammatory disease activity (Estess P, et al., 1998,Functional activation of lymphocyte CD44 in peripheral blood is a markerof autoimmune disease activity, J Clin Invest. 1998 Sep. 15;102(6):1173-82). Inflammatory bowel disease (IBD), including Crohn'sdisease (CD) and ulcerative colitis (UC), shares clinical andimmunological features with psoriasis. Genome-wide association studieshave found common susceptibility genes. However, epidemiologic dataevaluating the association between psoriasis, psoriatic arthritis andrisk of IBD are sparse. This research aimed to evaluate the associationbetween psoriasis, psoriatic arthritis and incident CD and UC amongwomen in the USA. Psoriasis with concomitant psoriatic arthritis isassociated with an increased risk of incident CD (Wen-Qing Li, 2013,Psoriasis, psoriatic arthritis and increased risk of incident Crohn'sdisease in US women, Ann Rheum Dis. 2013 July; 72(7):1200-5).Dermatomyositis (DM) is a connective-tissue disease related topolymyositis (PM) that is characterized by inflammation of the musclesand the skin. While DM most frequently affects the skin and muscles, itis a systemic disorder that may also affect the joints, the esophagus,the lungs, and, less commonly, the heart. Vasculitis is a group ofdisorders that destroy blood vessels by inflammation. Both arteries andveins are affected. The pathophysiology of vasculitis is not wellunderstood, but the ensuing inflammatory response has been generallywell described (Henry S. Su, 2012, Vasculitis: Molecular Imaging byTargeting the Inflammatory Enzyme Myeloperoxidase, Radiology, 2012January; 262(1):181-90). Behcet's disease (BD) is the only systemicvasculitis involving both arteries and vein in any sizes. It isfrequently encountered in rheumatology clinics. It has some majormorbidities and even fatal outcomes in some cases (M. B. Owlia, 2012,Behcet's Disease: New Concepts in Cardiovascular Involvements and FutureDirection for Treatment, ISRN Pharmacol, 2012: 760484).

Interferon alpha (IFNα) conjugated with polyethylene glycol (PEG) hasbeen widely used for the treatment of hepatitis C virus (HCV) infectionas a once-a-week injection formulation. However, the PEGylated IFNα hasa low efficacy of 39% and a side effect after repeated injectionspossibly due to the nonspecific delivery with PEGylation. Therefore,target specific long-acting hyaluronic acid-interferon alpha (HA-IFNα)conjugate was developed for the treatment of HCV infection. HA-IFNαconjugate was synthesized by coupling reaction between aldehyde modifiedHA and the N-terminal group of IFNα. The IFNα content could becontrolled in the range of 2-9 molecules per single HA chain with abioconjugation efficiency higher than 95%.

Several studies have implicated CD44 in direct interactions betweenbacteria and host cells, as well as in signaling events that alter hostcells and make them more susceptible to infection. Streptococcuspyogenes, for example, attaches to cells through its hyaluronan-richpolysaccharide capsule (or cell wall). HA binds to CD44, which in turntriggers tyrosine phosphorylation of several host-cell proteins, as wellas cytoskeletal rearrangements that cause ruffles and the extension oflamellipodia. As a result, the intercellular adhesion is loosened owingto a reduced number of tight junctions and of E-cadherin, a process thatallows the entry of bacteria into subepithelial tissue.

WO94/09811 describes the use of CD44 in treating inflammation ordetecting cancer metastasis. The authors show that CD44 is upregulatedin inflammatory conditions and CD44 peptides are capable of inhibitingT-cell activation. No data or claims are presented on inhibition ofmetastasis by CD44 and no claims are made towards use of CD44 forinhibiting tumor growth or angiogenesis. WO 99/45942 discloses the useof HA-binding proteins and peptides including CD44 to inhibit cancer andangiogenesis-dependent diseases. This publication uses metastatin, a 38kDa fragment of the cartilage link protein, as well as a HA-bindingpeptide derived from this fragment to inhibit pulmonary metastasis ofB16 mouse melanoma and Lewis lung carcinoma. In the case of theHA-binding peptide, growth of B16 melanoma on chicken CAM andendothelial cell migration on HA have been inhibited. In bothpublications the use of HA-binding peptides is directly related to theirability to bind hyaluronic acid.

U.S. Pat. No. 8,192,744 shows that soluble recombinant CD44 hyaluronicacid binding domain (CD44HABD) inhibits angiogenesis in vivo in chickand mouse and thereby inhibits human tumor growth of various origins.The invention discloses soluble non glycosylated CD44 recombinantproteins as a novel class of angiogenesis inhibitors based on targetingof vascular cell surface receptor.

Thus, the prior art discloses the potential use of CD44 to specify thatany effects are dependent on HA-CD44-interaction. Consequently, allutility ascribed this far to CD44-HA conjugate is directly dependent ontheir ability to bind hyaluronic acid.

However, some drugs are still not successfully conjugated onto HA andfurther experiments should be carried out to confirm the potentialusefulness of HA as site-delivery carrier of active compound. Inparticular, the prior art has not shown that the interactions betweenthe surface cell receptor CD44 and a conjugate of HA with an activecompound can be profitably exploited for a target delivery of suchactive compound in diseases characterized by an overexpression of CD44obtaining an effective therapeutic improvement of the same.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide new compound based onthe conjugation of HA with active compound suitable for a site deliveryof such active compound in diseases overexpressing the surface cellreceptor CD44.

The present invention, therefore, provides a compound conjugatingglycosaminoglycan with a drug, wherein the drug is used for treatingdiseases of inflammation that are highly related with the expression ofCD44.

In a first aspect, it is an object of the invention a compoundconsisting of a conjugate from a glycosaminoglycan and an activecompound, wherein the active compound is conjugated by means of afunctional group to a carboxylic group of the glycosaminoglycan, itsderivative, or a salt thereof to form a covalent conjugation, andwherein the active compound includes anti-inflammatory drug,anti-allergy drug and steroid.

The glycosaminoglycan of the conjugate according of the presentinvention is preferably hyaluronic acid.

Furthermore, the glycosaminoglycan conjugate according of the presentinvention is preferably for use for treating inflammation diseases.

Therefore, in a second aspect it is a further object of the inventionthe use of a compound consisting of a conjugate from a glycosaminoglycanand an active compound, wherein the active compound is conjugated bymeans of a functional group to a carboxylic group of theglycosaminoglycan, its derivative, or a salt thereof to form a covalentconjugation, and wherein the active compound consists of Celecoxib,Fexofenadine, Budesonide, and Prednisolone for the treatment ofinflammation and for the preparation of pharmaceutical compositions forsaid therapeutic treatment.

Yet in a further aspect, it is an object of the present invention themethod for preparing a compound consisting of a conjugate from aglycosaminoglycan and an active compound, wherein the active compound isconjugated by means of a functional group to a carboxylic group of theglycosaminoglycan, its derivative, or a salt thereof to form a covalentconjugation, and wherein the active compound consists of Celecoxib,Fexofenadine, Budesonide, and Prednisolone.

BRIEF DESCRIPTION OF THE DRAWINGS

To adequately describe the present invention, references to embodimentsthereof are illustrated in the appended drawings. These drawingsherewith form a part of the specification. However, the appendeddrawings are not to be considered limiting in their scope.

FIG. 1 shows the affinity of hyaluronic acids (HAs) by fluorescent indexin normal and injured colon tissues;

FIG. 2 shows the fluorescence results of HA-dye compound working on HCT15 cell line and HT29 cell line with different time course, wherein FIG.2A represents HCT 15 cell line at 6 hours; FIG. 2B represents HCT 15cell line at 12 hours; FIG. 2C represents HT29 cell line at 6 hours;FIG. 2D represents HT29 cell line at 12 hours.

FIG. 3 shows the structure of HA-Celecoxib conjugate.

FIG. 4 shows the anti-inflammation effect of Control, lipopolysaccharide(LPS), HA, Celecoxib (Cele), and HA-Celecoxib conjugate (HA-cele) on Raw264.7 cell line. The inflammation index herein is change percentage ofprostaglandin E₂ (PGE₂) amount.

FIG. 5 shows the structure of HA-ADH-Budesonide conjugate.

FIG. 6 shows the anti-inflammation effect of Control (ctrl), LPS, HA, HAplus LPS (HA+LPS), Budesonide (bude.), Budesonide plus LPS (bude+LPS),HA-Budesonide conjugate (HA-bude, “HA-bude” stands for“HA-ADH-Budesonide”) and HA-Budesonide conjugate plus LPS (HA-bude+LPS)on Raw 264.7 cell line. The inflammation index herein is change ofnitrite (NO) concentration.

FIG. 7 shows the structure of HA-ADH-Fexofenadine conjugate.

FIG. 8 shows the anti-inflammation effect of Control, LPS, HA,Fexofenadine (Fexo), and HA-Fexofenadine conjugate (HA-fexo, “HA-fexo”stands for “HA-ADH-Fexofenadine”) on Raw 264.7 cell line. Theinflammation index herein is change percentage of prostaglandin E₂(PGE₂) amount.

FIG. 9 shows the structure of HA-ADH-Prednisolone conjugate.

FIG. 10 shows the anti-inflammation effect of Control, LPS, HA,Prednisolone (Pred), and HA-Prednisolone conjugate (HA-pred, “HA-pred”stands for “HA-ADH-Prednisolone”) on Raw 264.7 cell line. Theinflammation index herein is change percentage of prostaglandin E₂(PGE₂) amount.

FIG. 11 shows treatment effect of Rheumatoid arthritis (RA) by control(vehicle), Prednisolone (Pred), HA conjugated with Prednisolone(HA-ADH-Pred), and HA. The treatment index herein is change percentageof paw thickness (mean±SE). Rats were fed for inducing RA during D1 toD13, and dosing on D14. Data were calculated by Non-parametricstatistics, *p<0.05 vs. Vehicle group; ^(#)p<0.05 vs. Prep group and^(&)p<0.05 vs. HA group.

FIG. 12 shows treatment effect of Rheumatoid arthritis (RA) by control(vehicle), Prednisolone (Pred), HA conjugated with Prednisolone(HA-ADH-Pred), and HA. The treatment index herein is change percentageof ankle circumference (mean±SE). Rats were fed for inducing RA duringD1 to D13, and dosing on D14. Data were calculated by Non-parametricstatistics, *p<0.05 vs. Vehicle group; ^(#)p<0.05 vs. Prep group and^(&)p<0.05 vs. HA group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Other objectives, advantages and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

In general, a drug orally administered or injected into circulationsystem must directly arrive at its targeted treatment area, andtherefore the drug effect on targeted disease and normal organ is verysimilar because the concentration and specificity are not so high on thetargeted site, limited by safety profile.

In order to improve the therapy efficacy combining the same with goodsafety profile, one strategy is to modify the drug to be moretarget-selective to the disease area through a covalent binding the drugwith a carrier. This is a need particularly felt in the field ofanti-inflammatory therapy as previously anticipated.

At this purpose, the inventor has conceived the idea to exploit theinteractions between the hyaluronic acid (HA) and its receptor CD44 fora target delivering active substance.

The idea to maintain the relative higher concentration of the drug onthe targeted site versus normal tissue or organ has been established bythe inventor following long-term study and experiment on HA.

The results, from which the present invention originates, are fullydescribed in the examples and are hereinafter briefly summarized.

In fact, the present invention finds support on the result showing thathyaluronic acids having different average molecular weights (MW) have anadhesion index higher in injured tissue than in normal tissue and thatthe HA with low average molecular weight performs better than the HAswith high average molecular weights. In particular, as shown in FIG. 1,comparing the differences among HAs of three average molecular weightsadhered on the injured colon tissues, the fluorescent index of adhesionof 350 KDa HA by the injured colon tissues was higher than the HAs ofthe other two average molecular weights (2000 KDa=2 MDa) and (1000 KDa=1MDa). Further, the fluorescent index of adhesion of 1 MDa HA by evennormal or injured colon tissues was higher than 2 MDa HA. This resultconfirmed that the HA can more specifically adhere on the inflammationsite, which induces the inventor to further invent the present inventionand to verify whether this peculiar feature of tissue adhesion ofhyaluronic acid, allegedly due to an interaction of HA with its surfacecell receptor CD44, can be maintain when this glycosaminoglycan isconjugated with other compounds. Therefore, the inventor furtherconjugates a drug with HA in order to verify if HA can be used as atargeted delivery vehicle to conduct the drug onto CD44 abundance site.As aforementioned, when CD44 is overexpressed during the situation ofexistence of inflammation, infection or a cancer, a related drug caneasily arrive at and retain relative high concentration on the targetedsite owing to ligand HA attaches onto receptor CD44. In accompany withHA's adherence effect to inflammation site or CD44 abundance site, theconjugated drug should especially aggregate on the targeted part toenhance the therapy efficacy owing to relative higher concentration ofthe drug on the site, and hence decreasing accordingly the amount of thedrug utilized with better safety profile. In order to confirm the drugor dye has been successfully conjugated with HA and further confirm theHA attachment effect, the inventor of the present invention conducted anexperiment including conjugating dye onto HA (HA-dye) and treating withthe cell lines and mice separately. FIG. 2A and FIG. 2B show theexperiments at different working times on cell line HCT15 (a colorectaladenocarcinoma with less CD44), and FIG. 2C and FIG. 2D show theexperiments at different working times on cell line HT29 (a colorectaladenocarcinoma with rich CD44). The results of HT29 (FIG. 2C and FIG.2D) show the HA-dye have been successfully conjugated and attached ontoCD44 abundant area of HT29 (FIG. 2C), and even enter into HT29 cells(FIG. 2D). That means the idea of the present invention is proper andeffective and also means drug or dye can be conjugated to HA and that HAretain its capability to bind CD44.

The attachment condition of free dye and HA-dye on cell lines of HT29and HCT15 of mice for 4 weeks was conducted. The free dye was injectedinto the tail vein of the mice. The result showed that the two differentCD44 expression cancer cells without any difference in attachmentresult. The ratio of attachment area of HT29 is 50.15%, whereas HCT15 is49.86%. However, when the HA conjugated dye was injected into the micetail vein, the more CD44 expression cancer cell HT 29 showed significantconcentration of HA conjugated dye, but the less CD44 expression HCT15showed very limited result. The ratio of attachment area of HT29 is74.15%, whereas HCT15 is 25.85%. The result can show that when dyeconjugated with HA, the concentration of dye was increased owing to HAattached on CD44 abundant site.

CD44 highly related diseases include cancer, infection and inflammation.Infectious pathogens include, but not limited to, some viruses,bacteria, fungi, protozoa, multicellular parasites, and aberrantproteins known as prions. These pathogens are the cause of diseaseepidemics, in the sense that without the pathogen, no infectiousepidemic occurs. In a preferred embodiment, infection disease includeslower respiratory infections, HIV/AIDS, diarrheal diseases,tuberculosis, malaria, measles, pertussis, tetanus, meningitis,syphilis, hepatitis B, sepsis, and tropical diseases. Inflammatoryabnormalities are a large group of disorders which underlie a vastvariety of human diseases. In a preferred embodiment, inflammationdisease includes acne vulgaris, allergic diseases, asthma, autoimmunediseases, celiac disease, chronic prostatitis, glomerulonephritis,hypersensitivities, inflammatory bowel diseases, pelvic, inflammatorydisease, reperfusion injury, rheumatoid arthritis, sarcoidosis,transplant rejection, vasculitis, and interstitial cystitis.

In the specification and in the claims the term “drug” or “compound” or“agent” for use in the present invention may comprise anti-asthma drug,anti-histamine drug, anti-infection drug, anti-inflammatory drug,anti-allergy drug, anti-virus drug, immunosuppressant, NSAID(Non-Steroidal Anti-Inflammatory Drug), and steroid. Among them,anti-allergy drug, anti-infection drug, anti-inflammatory drug, andsteroid are preferred. The majority of anti-allergy drugs can be dividedinto anti-histamines, anti-inflammatory agents, decongestants andsteroid such as Fexofenadine, cetirizine, chlorpheniramine maleate,pseudoephedrine, and Budesonide. The majority of anti-infection drugscan be divided into local anti-infection means that are used externallyon skin or mucous membranes without entering the blood stream andmetabolizing in the liver; and systemic anti-infection means which areused to treat internal organ infections through oral or injectedadministration. The majority of anti-inflammatory drugs can be dividedinto non-steroidal anti-inflammatories or NSAIDs, COX-2 antagonist andsteroid.

In another embodiment, examples of appropriate drugs are anti-asthmadrug, anti-fungal drug, anti-histamine drug, anti-inflammatory drug,anti-virus drug, NSAID and steroid thereof. In a preferred embodiment,the conjugation of anti-asthma drug including Salbutamol; NSAIDincluding Nimesulide, Celecoxib, Meloxicam, Diclofenac and Piroxicam;anti-allergy drug including Fexofenadine; anti-fungal drug includingAmphotericin B; anti-virus drug including Ribavarin; and steroidincluding Budesonide and Prednisolone to HA were preferred. Further,COX-2 antagonist includes Celecoxib.

The aim of the present invention is binding or conjugating HA with adrug aforesaid, with or without a linker or spacer, by carboxyl group,hydroxyl group, or amino group of HA to accomplish working effect onspecific location and specific time. Therefore, HA as a target deliveryvehicle to carry the drug to the specific site that has abundant CD44can produce better treatment efficacy and safety.

As used herein, in general, the term “linker” or “spacer” means anorganic moiety that connects two parts of a compound. Linkers typicallycomprise a direct bond or an atom such as oxygen or sulfur, a unit suchas SS, NH, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such assubstituted or unsubstituted alkyl where one or more methylenes can beinterrupted or terminated by O, S, S(O), SO₂, NH, NH₂, C(O). The term“linker” or “spacer” of the present invention may be absent and denotesany chemical compound present between the drug and the HA which may beremoved chemically, enzymatically or may decompose spontaneously; italso contains at least one other group useful for linking the drug, e.g.amino, thiol, further carboxyl groups, etc. The linker or spacer may bea polypeptide, a peptide, or a lipid.

Suitable linkers or spacers are e.g. adipic dihydrazide (ADH), linear orbranched, aliphatic, aromatic or araliphatic C₂-C₂₀ dicarboxylic acids,amino acids, peptides.

The role of the linker, whenever present, consists in creating an arm ora spacer between the hyaluronic acid and the drug. The linker engages,on one side, the HA via the amide, carboxyl group, hydroxyl group, oramino group linkage and, on the other side, the drug via any possiblecovalent-type bond.

When the linker or spacer is a dicarboxylic acid, the carboxylic groupforming the ester bond with the drug may be the hydroxyl group of thecompound. When the linker or spacer is a dihydrazide, the amino groupforming the amide bond with HA may be the free carboxylic group of theHA. Preferred linkers or spacers are: succinic acid to drug, adipicdihydrazide to HA.

In the preferred embodiment, the present invention provides a compoundconsisting of a conjugate from a glycosaminoglycan, preferablyhyaluronic acid, and an active compound, wherein the active compound isconjugated by means of a functional group to a carboxylic group of theglycosaminoglycan, its derivative, or a salt thereof to form a covalentconjugation, and wherein the active compound consists ofanti-inflammatory drug, anti-allergy drug and steroid.

The active compounds anti-inflammatory drug including Celecoxib which isa COX-2 antagonist, anti-allergy drug including Fexofenadine, and thesteroid including Budesonide and Prednisolone can be bound preferablydirectly or indirectly through a linker by means the functionalcarboxylic group of the HA and the —NH₂ group of the active compounds.

In a preferred embodiment of the present invention, the covalentconjugation, either direct or indirect through a linker, between one ofthe functional carboxyl groups of HA and of the active compound can beeither an amidic bond or an ester bond.

In case of indirect conjugation by means of a linker, said linkers areselected from a dihydrazide, adipic dihydrazide, polypeptide, a peptide,a lipid, an aminoacid or a linear or branched, aliphatic, aromatic oraraliphatic C₂-C₂₀ dicarboxylic acids.

The preferred HA for conjugation has an average molecular weight in therange comprised from 5 kDa to 2000 kDa and the conjugation involves atleast 40% of the carboxyl group of HA.

In order to treat the acute or chronic inflammation, where the acute orchronic inflammation is induced or caused by infection, injury,autoimmune disease, or allergy and the autoimmune disease includingRheumatoid arthritis, the preferred embodiment of the formulation ordosage form of the present invention including an excipient and/ordiluent to formulate an administrating dosage form for oral orcirculation system use. The more preferred embodiment of the oral dosageform is selected from the group consisting of solid dosage form,solution including, but not limited to suspension, tablet including, butnot limited to controlled-release tablet, and capsule including, but notlimited to enteric-coated capsule. The more preferred embodiment of thecirculation system or systemic administration form is selected from thegroup consisting of intro-venous (IV), intra-muscle (IM) andsubcutaneous (SC).

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE Example 1 The Adhesion of HA in Colon Tissue (IVIS ImageSystem-Vision 3)

Procedure:

1. 0.25 g high molecule weight sodium hyaluronate powder (HHA; Mw: 2MDa; Freda) and 0.25 g low molecule weight sodium hyaluronate powder(LHA; Mw: 0.35 MDa; Freda) were added into 50 ml PBS buffer (Phosphatebuffered saline) respectively to form 0.5% solution, and then stirredfor 6 hours until the powder was totally dissolved. 0.25 g mediummolecule weight sodium hyaluronate powder (MHA; Mw: 1 MDa; Freda) wasadded into 50 ml PBS buffer, and then stirred for 6 hours until thepowder was totally dissolved and ready for use in the following steps.

2. Fluorescent HA (HA-f) was prepared by (1) 0.39 g MES free acid(2-(N-morpholino) ethanesulfonic acid, Calbiochem) and was dissolved in100 ml dd water. (2) Solution A: 65 mg fluroresceinamine powder, (isomerI, Fluka) was dissolved in 9 ml 95% EtOH solution and then stirred for10 minutes under a condition that light was prohibited. (3) Solution B:359 mg EDC powder (N-(3-Dimethylamino propyl)-N-ethyl carbodiimidehydrochloride, Sigma) was dissolved in 9 ml MES buffer and then stirredfor 10 minutes. (4) Solution C: 216 mg NHS powder (N-Hydroxysuccinimde,Sigma) was dissolved in 9 ml MES buffer and then stirred for 10 minutes.(5) 3 ml Solution A was slowly dropped into 50 ml 0.5% HA solution andthen stirred for 10 minutes under a condition that light was prohibited.(6) 3 ml Solution B and 5 ml Solution C were separately dropped into thesolution of step (5) and then stirred for 10 minutes under a conditionthat light was prohibited. (7) 0.02 M MES buffer was slowly added intothe solution of step (6) until the volume reached 100 ml and was thenstirred for 24 hours at room temperature under a condition that lightwas prohibited. (8) The product after reaction was poured into adialysis tubing (MW: 12000˜14000) in 5 L dd water as a dialysis solutionand then stirred for 5 days at 4° C. under a condition that light wasprohibited with dialysis solution being changed every 12 hours until thedialysis solution had no fluorescence. (9) The liquid after dialysis wasallocated into 50 c.c. plastic centrifuge tubes and then reserved at−20° C. refrigerator overnight, followed by drying in a freeze-dryingmachine under a condition that light was prohibited. (10) The dried HA-fpowder was reserved at −20° C. refrigerator. (11) 50 mg HA-f powder wasslowly added into 10 ml PBS buffer and then stirred for 6 hours untilthe powder was totally dissolved.

3. Colon tissue of SD-rat (Sprague-Dawley Rat) aged 7-8 weeks was cut byscalpel and then washed by PBS buffer, followed by being cut to 3-4 cmlong with soaking in PBS buffer finally.

4. Injured colon tissue was prepared by brushing by toothbrush for 20times longitudinally and then soaking in PBS buffer.

5. Normal and injured colon tissues were put into a 12-well plate andthen 1 ml 0.5% HA-f solution was added into each well and shaken for 2hours at room temperature. Surplus HA-f solution was sucked by tip 2hours later, and then soaked into PBS buffer for 10 minutes followed byremoving PBS buffer repeatedly for 3 times.

6. Cleaned colon tissue was placed in a 12-well plate with lining tissueupwards and then placed onto the dock of the IVIS (in vivo image system,XENOGEN). The default parameter was set up as GFP (green fluorescentprotein) whereas the excitation was 465 nm and the emission was 500 nmand then the image was captured by software.

7. All values are calculated as means of n observations. Thehistological index was analyzed by Student's t-test.

Result: The fluorescent index was quantified and arranged as in FIG. 1.The fluorescent index of normal colon tissue was defined as 1. The othercolon tissues tests were calibrated by the defined value. The resultshowed that the HAs with the same average Mw were adhered in the injuredcolon tissues with obviously higher fluorescent index than in the normalcolon tissues (P<0.01). Comparing the difference between HAs of threedifferent average molecular weights adhered in the injured colontissues, the fluorescent index of adhesion of 350 KDa HA by the injuredcolon tissues was obviously higher than HAs of the other two averagemolecular weights (2 MDa and 1 MDa). Further, the fluorescent index ofadhesion of 1 MDa HA by even normal or injured colon tissues was higherthan 2 MDa HA.

Example 2 HA-Dye Conjugation Process and HA-Dye In Vitro Image

The following whole process of HA-dye conjugation must be kept in dark.The synthesis of HA-ADH

1. HA (0.34 MDa, 50 mg) was dissolved in water to give a concentrationof 4 mg/ml.

2. 5-fold excess (114.8 mg) of ADH was added into the solution.

3. The pH of the reaction mixture was adjusted to 4.75 by addition of0.1 N HCl.

4. Next, 1 equiv. (25.1 mg) of EDC was added in solid form. The pH ofthe reaction mixture was maintained at 4.75 by addition of 0.1 N HCl.

5. After 15 minutes reacting, the reaction was quenched by addition of0.1 N NaOH to adjust the pH of reaction mixture to 7.0.

6. The reaction mixture was then transferred to pretreated dialysistubing (Mw cutoff 3500) and dialyzed exhaustively against 100 mM NaCl,then 25% EtOH/water 4 cycles and finally water. The solution was thenfiltered through 0.2 μm cellulose acetate membrane, flash frozen, andlyophilized.

7. The substitution degree of ADH was measured by 1 H NMR. The synthesisof HA-ADH-FITC

1. HA-ADH (DS=36%) 88 mg was dissolved in 35 ml water

2. FITC 9.5 mg was dissolved in 10 ml DMSO.

3. Mix HA-ADH solution and FITC solution

4. After stirred 48 h at room temperature, the solution was dialyzed 3days with 0.3 M NaCl and pure water alternately using MWCO 12000-14000dialysis bag.

5. The solution was then freeze-drying 2 days.

6. Finally the degree of substitution was determined by UV spectrum.

HA-Dye In Vitro Image

(1) 1×10⁵ HT 29 cells and HCT15 cells (human colon carcinoma, a CD44positive cell) were seeded onto a microscope slide in a 3.5 cm dish.

(2) Indicated dye concentrations, 1 μM of HA-dye (HA: 0.34 MDa) wereadded into cells for indicated time respectively.

(3) After incubation, cells were washed in PBS, and then fixed in 3.7%formaldehyde.

(4) Observation of the interaction between HA-dye and cells wasperformed by confocal microscopy.

Result: The fluorescent view can show the attachment site and amount ofdye on HCT15 (FIG. 2A and FIG. 2B) and HT29 (FIG. 2C and FIG. 2D). Theresults reveal that dye has been successfully conjugated with HA and HAenhances HA-dye concentration on CD44 abundant site on HT29, whereasHT29 has stronger fluorescence that meets with more abundant CD44 thanHCT15 has. Even proved that HA-dye can enter the cells (FIG. 2D). TheHA-dye was accumulated after a 6 hours treatment and internalized aftera 12 hours treatment in HT29 (more CD44). Such phenomenon was notobserved in HCT15 (less CD44) after a 6 hours or a 12 hours treatment ofHA-dye.

Example 3 Synthesis of HA-Celecoxib Conjugate Procedure

1. 100 mg HA (10K-700 KDa) were dissolved in 25 ml DD water.

2. Tetrabutylammonium hydroxide (TBA-OH) 0.8 eq was added into HAsolution and stirred for 16 hours.

3. Dried the solution and the HA-TBA white solid was acquired.

4. HA-TBA 40 mg was dissolved in 1 ml DD water and then EDC 30 mg andNHS powder 18 mg were added into the solution and stirred at roomtemperature for 5 minutes.

5. Celecoxib 4 mg was dissolved in 2 ml dimethylsulfoxide (DMSO)solution.

6. This mixture (HA-TBA, EDC, NHS and Celecoxib) was stirred at roomtemperature for 72 hours.

7. The mixture was dialyzed for 1 day against that a ratio of DMSO andDD water is 2 to 1 by using dialyzer bag (MWCO: 1200˜1400) and changedthe solution three times.

8. The mixture was then dialyzed for 2 days against 0.3 M NaCl by usingdialyzer bag (MWCO: 1200˜1400) and changed the solution two times a day.

9. HA-Celecoxib powder was acquired by dehydration through freeze dryerfrom HA-Celecoxib solution.

Result: FIG. 3 shows the structure of HA-Celecoxib conjugate.

Example 4 In Vitro Anti-Inflammatory Effects of HA-Celecoxib on RAW264.7 Cells Procedure

1. Raw 264.7 cells (1×10⁶ cells/well, 1000 μL) were incubated in a24-well culture plate in the presence of 5% CO₂ at 37° C. for 24 hours.

2. The medium was changed to DMEM containing 10% FBS, and the cells weretreated with various concentrations of compounds (100 nM Celecoxib,HA-Celecoxib (equal to 100 nM Celecoxib), and 4.9 μg/ml HA) for 4 hoursfollowed by 1 μg/mL of lipopolysaccharide (LPS) treatment for 24 h.

3. Cell media were collected, and used for measuring PGE₂.

4. Nunc-Immuno 96-well plates were coated with goat polyclonalanti-mouse IgG secondary antibody.

5. Aliquots of cell media were added to the immune plate with primaryPGE₂ monoclonal antibody and a tracer PGE₂-acetylcholinesteraseovernight at room temperature in the dark.

6. The next day, the wells were aspirated, washed with 100 μL washbuffer (Cayman Chemical, Ann Arbor, Mich., USA) five times.

7. 200 μL of Elman's reagent was added in each well, and incubated 60-90minutes at room temperature out of direct light.

8. Gentle rotating/shaking was used to decrease the time required forcolor development.

9. Absorbance was read at 412 nm.

10. The PGE₂ concentration of each sample was calculated from a PGE₂standard curve.

11. The PGE₂ amount percentage was as a function of the PGE₂ level ofeach sample divided by the one of the control group which wasLPS-treated, then multiplied this value by 100.

12. The actual amount of PGE₂ was estimated to be 1370 ng/ml for thecontrol group which was LPS-treated.

Result: As shown in FIG. 4, inflammation was successfully induced by LPSas the index is the amount of PGE₂. Drug-only group (Celecoxib) hastreatment effect; however, when HA conjugated with Celecoxib (HA-cele),it has better treatment effect than drug only.

Example 5 Synthesis of HA-ADH-Budesonide Conjugate Procedure Synthesisof HA-ADH:

1. HA (50 mg) was dissolved in water to give a concentration of 4 mg/ml.

2. 5-fold excess (114.8 mg) of ADH was added into the solution.

3. The pH of the reaction mixture was adjusted to 4.75 by addition of0.1 N HCl.

4. Next, 1 equiv. (25.1 mg) of EDC was added in solid form. The pH ofthe reaction mixture was maintained at 4.75 by addition of 0.1 N HCl.

5. After 15 minutes reacting, the reaction was quenched by addition of0.1 N NaOH to adjust the pH of reaction mixture to 7.0.

6. The reaction mixture was then transferred to pretreated dialysistubing (Mw cutoff 3500) and dialyzed exhaustively against 100 mM NaCl,then 25% EtOH/water 4 cycles and finally water. The solution was thenfiltered through 0.2 μm cellulose acetate membrane, flash frozen, andlyophilized.

7. The substitution degree of ADH was measured by 1 H NMR.

Synthesis of HA-ADH-Budesonide:

1. HA-ADH-Budesonide succinate was formed by chemical conjugation of thecarboxyl group of Budesonide succinate with the amine group of HA-ADHusing EDC and NHS.

2. 7.4 mg Budesonide succinate was dissolved in 1 ml DMSO and reactedwith 1 ml pure water containing EDC 14.5 μmol and NHS 14.5 μmol for 5minutes.

3. After 5 minutes, the mixed solution was added dropwise into 8 mlWater: DMSO=1:1(v/v) cosolvent containing 20 mg HA-ADH

4. The mixed solution was stirred 24 hours at room temperature (about30° C.).

5. After 24 hours, the solution was dialyzed 3 days with 0.3 M NaCl andpure water alternately using MWCO 12000-14000 dialysis bag.

6. The solution was then freeze-drying 2 days and stored at 4° C.

7. Finally the degree of substitution was determined by 1 H-NMR.

Result: FIG. 5 shows the structure of HA-ADH-Budesonide conjugate.

Example 6 In Vitro Anti-Inflammatory Effects of HA-ADH-Budesonide on RAW264.7 Cells Procedure

1. Raw 264.7 cells were suspended in culture medium without phenol red(Gibco-BRL, Vienna, Austria) and adjusted to 10⁶ cells/ml, and treatedwith 10 μM Budesonide, 378 μg/ml HA and HA-ADH-Budesonide (equal to 10μM Budesonide).

2. After treatment with drugs for 4 hours, 1 μg/mL LPS was added intomedium for 24 hours.

3. Cells treated with medium only served as a negative control group andtreated with LPS (1 μg/mL) as a positive control group.

4. The culture supernatants were removed subsequently and the nitricoxide were accumulated for the measurement of iNOS activity by theGriess-reaction.

5. 100 μl Griess reagent (1% sulfanilamide, 0.1% naphthlethylenediaminedihydrochloride in 2.5% phosphoric acid) was added into 100 μl culturesupernatant and the color development was assessed at 550 nm with aELISA microplate reader (SpectraMax® M2e Multimode Plate Reader,Molecular Devices, USA).

6. Standard curves were generated with a serial dilution of sodiumnitrite dissolved in culture medium which is phenol red free.

Result: As shown in FIG. 6, when comparing with groups LPS-only, HA+LPS,bude.+LPS, and HA-bude+LPS (“HA-bude” stands for“HA-ADH-Budesonide”), groups HA+LPS and HA-bude+LPS have treatmenteffect. The inflammation index herein is nitrite.

Example 7 Synthesis of HA-ADH-Fexofenadine Conjugate Procedure

1. HA-ADH-Fexofenadine was synthesized by chemical conjugation of thecarboxyl group of Fexofenadine with the amine group of HA-ADH using EDCand NHS.

2. 7.8 mg Fexofenadine was dissolved in 1 ml DMSO and reacted with 1 mlpure water containing 73.3 μmol EDC and 73.9 μmol NHS for 5 minutes.

3. After 5 minutes, the mixed solution was added dropwise into 8 mlwater: DMSO=1:1(v/v) cosolvent containing 20 mg HA-ADH

4. The mixed solution was stirred for 24 hours at room temperature(about 30° C.).

5. After 24 hours, the solution was dialyzed 3 days with 0.3 M NaCl andwater alternately using MWCO 12000-14000 dialysis bag serially.

6. The solution was then freeze-drying 2 days and stored at 4° C.

7. Finally the degree of substitution was determined by 1H-NMR.

Result: FIG. 7 shows the structure of HA-ADH-Fexofenadine conjugate.

Example 8 In Vitro Anti-Inflammatory Effects of HA-ADH-Fexofenadine onRAW 264.7 Cells Procedure

1. Raw 264.7 cells (1×10⁶ cells/well, 1000 μL) were incubated in a24-well culture plate in the presence of 5% CO₂ at 37° C. for 24 hours.

2. The medium was changed to DMEM containing 10% FBS, and the cells weretreated with various concentrations of compounds (100 μM Fexofenadine,HA-ADH-Fexofenadine (equal to 100 μM Fexofenadine), and 2.96 mg/ml HA)for 4 hours followed by 1 μg/mL of LPS treatment for 24 h.

3. Cell media were collected, and used for measuring PGE₂.

4. Nunc-Immuno 96-well plates were coated with goat polyclonalanti-mouse IgG secondary antibody.

5. Aliquots of cell media were added to the immune plate with primaryPGE₂ monoclonal antibody and a tracer PGE₂-acetylcholinesteraseovernight at room temperature in the dark.

6. The next day, the wells were aspirated, washed with 100 μL washbuffer five times.

7. 200 μL of Elman's reagent was added in each well, and incubated 60-90minutes at room temperature out of direct light.

8. Gentle rotating/shaking was used to decrease the time required forcolor development.

9. Absorbance was read at 412 nm.

10. The PGE₂ concentration of each sample was calculated from a PGE₂standard curve.

11. The PGE₂ amount percentage was as a function of the PGE₂ level ofeach sample divided by the one of the control group which wasLPS-treated, then multiplied this value by 100.

12. The actual amount of PGE₂ was estimated to be 1370 ng/ml for thecontrol group which was LPS-treated.

Result: As shown in FIG. 8, drug-only group (Fexofenadine) has treatmenteffect; however, when HA conjugated with Fexofenadine (HA-fexo,“HA-fexo” stands for “HA-ADH-Fexofenadine”), it has better treatmenteffect than drug only.

Example 9 Synthesis of HA-ADH-Prednisolone Conjugate Procedure

1. Synthesis of Prednisolone 21-hemiester:

2. A solution of Prednisolone (1 g; 2.77 mmol) and succinic anhydride(1.125 g; 12.55 mmol) in pyridine (8 mL) was stirred at roomtemperature.

3. After 24 h, the reaction mixture was poured onto a mixture of ice (25g), water (25 mL) and conc. HCl (10 mL).

4. The separated crystals were collected by filtration, washed withwater, dried, recrystallized from toluene and dried overnight again.

5. The prepared hemiester was characterized by 1H-NMR spectroscopy.

Synthesis of HA-ADH-Prednisolone Hemiester:

1. SOLUTION 1: EDC 73.3 μmol (14 mg) and NHS 73.9 μmol (8.5 mg) wereadded in 1 ml water.

2. SOLUTION 2: Prednisolone hemiester 14.5 μmol (6.4 mg) was dissolvedin 1 ml DMSO.

3. SOLUTION 3: HA-ADH (D.S=31%) 20 mg (ADH:14.5 μmol) was dissolved in 8ml cosolvent (water: DMSO=1:1).

4. Mix SOLUTION 1 & 2, and stir 5 minutes to activate carbonyl group onPrednisolone hemiester.

5. After 5 minutes, the mixture was added dropwise (1 ml/min) toSOLUTION 3 under stirring mixing.

6. The mixed solution was stirred 24 hours at room temperature (about30° C.).

7. After 24 hours, the solution was dialyzed 3 days with 0.3 M NaCl/purewater alternately using MWCO 12000-14000 dialysis bag.

8. The solution was then freeze-drying 2 days.

9. Finally the degree of substitution of HA-ADH-Pred was determined by1H-NMR.

Result: FIG. 9 shows the structure of HA-ADH-Prednisolone conjugate.

Example 10 In Vitro Anti-Inflammatory Effects of HA-ADH-Prednisolone onRAW 264.7 Cells

1. Raw 264.7 cells (1×10⁶ cells/well, 1000 μL) were incubated in a24-well culture plate in the presence of 5% CO₂ at 37° C. for 24 hours.

2. The medium was changed to DMEM containing 10% FBS, and the cells weretreated with various concentrations of compounds (86.7 μM Prednisolone,HA-ADH-Prednisolone (equal to 86.7 μM Prednisolone), and 70 μg/ml HA)for 4 hours followed by 1 μg/mL of LPS treatment for 24 hours.

3. Cell media were collected, and used for measuring PGE₂.

4. Nunc-Immuno 96-well plates were coated with goat polyclonalanti-mouse IgG secondary antibody.

5. Aliquots of cell media were added to the immune plate with primaryPGE₂ monoclonal antibody and a tracer PGE₂-acetylcholinesteraseovernight at room temperature in the dark.

6. The next day, the wells were aspirated, washed with 100 μL washbuffer five times.

7. 200 μL of Elman's reagent was added in each well, and incubated 60-90minutes at room temperature out of direct light.

8. Gentle rotating/shaking was used to decrease the time required forcolor development.

9. Absorbance was read at 412 nm.

10. The PGE₂ concentration of each sample was calculated from a PGE₂standard curve.

11. The PGE₂ amount percentage was as a function of the PGE₂ level ofeach sample divided by the one of the control group which wasLPS-treated, then multiplied this value by 100.

12. The actual amount of PGE₂ was estimated to be 1370 ng/ml for thecontrol group which was LPS-treated.

Result: As shown in FIG. 10, drug-only group (Prednisolone) hastreatment effect; however, when HA conjugated with Prednisolone(HA-pred, “HA-pred” stands for “HA-ADH-Prednisolone”), it has bettertreatment effect than drug only.

Example 11 In Vivo Treatment Effect for Rheumatoid Arthritis (RA) byHA-ADH-Prednisolone Procedure

1. Thirty of eight weeks male Sprague-Dawely rats (BioLASCO Taiwan Co.,Ltd.) were prepared. Rats were divided into 4 groups randomly (n=9 forvehicle and Prednisolone group, n=6 for HA-ADH-Prednisolone and HAgroup). Every two rats were placed into one cage in rodent animalfacility in Institute of Taiwan Animal Technology.

2. Inject 0.1 mL of CFA (Complete Freund's Adjuvant, 10 mg/mL, ChondrexInc.) containing 10 mg/mL of heat-killed mycobacterium intosubcutaneously at a footpad of right hind limb.

3. The needle should be inserted just under the skin of the footpadpointing toward the ankle: this maximizes delivery of the adjuvant tothe draining popliteal lymph nodes.

4. Severe and acute inflammation is observed within 30 minutes ofinjection, peaks within 3 to 4 days, and often persists for 20 to 25days.

5. Rat were treated at day 14 after induction of the Adjuvant-InducedArthritis (AIA) by intravenous injection of Prednisolone orHA-Prednisolone (both 10 mg/kg), or saline as a control.

6. Rats were sacrificed at day 20 after treatment and tissues wereisolated hereafter.

Arthritis Measurements:

-   -   Paw thickness and Ankle circumference: Paw thickness determined        by measuring paw diameter using a caliper, such as Mitsutoyo        digimatic caliper. Ankle circumference determined by measuring 2        perpendicular diameters, the latero-lateral diameter (a) and the        anteroposterior diameter (b), with a digital caliper and using        the following formula: circumference=2π(√{square root over        ((a²+b²)/2)})        Result: As shown in FIG. 11, RA has been successfully induced.        At the beginning of the date administering drug (day 14),        HA-only group has almost the same trend comparing with control        group (vehicle) in the kind of index as change of paw thickness.        The drug-only group (Prednisolone) showed the treatment effect.        However, when drug conjugated with HA (HA-ADH-Pred group), its        treatment effect was even better than the drug only, and has        statistically significance. Also, as shown in FIG. 12, HA only        even enhance RA symptom in this kind of index (change of ankle        circumference); however, when drug conjugated with HA        (HA-ADH-Pred group), its treatment effect was better than the        drug only, and has statistically significance.

What is claimed is:
 1. A compound consisting of a conjugate from aglycosaminoglycan and an active compound, wherein the active compound isconjugated by means of a functional group to a carboxylic group of theglycosaminoglycan, its derivative, or a salt thereof to form a covalentconjugation, and wherein the active compound consists ofanti-inflammatory drug, anti-allergy drug and steroid.
 2. The compoundaccording to claim 1, wherein the anti-inflammatory drug is COX-2antagonist.
 3. The compound according to claim 2, wherein the COX-2antagonist is Celecoxib.
 4. The compound according to claim 1, whereinthe anti-allergy drug is Fexofenadine.
 5. The compound according toclaim 1, wherein the steroid is Budesonide and Prednisolone.
 6. Thecompound according to claim 1, wherein the covalent conjugation is adirect conjugation by means of an amide bond or an ester bond.
 7. Thecompound according to claim 1, wherein the active compound is alsoindirectly conjugated to the carboxylic group of the glycosaminoglycanthrough a linker.
 8. The compound according to claim 7, wherein thelinker is selected from the group consisting of adipic dihydrazide,polypeptide, peptide, lipid, amino acid and linear or branched,aliphatic, aromatic or araliphatic C₂-C₂₀ dicarboxylic acids.
 9. Thecompound according to claim 1, wherein the glycosaminoglycan ishyaluronic acid.
 10. The compound according to claim 9, wherein thehyaluronic acid has an average molecular weight comprised in the rangefrom 5 kDa to 2000 kDa.
 11. The compound according to claim 1, for usein the treatment of acute or chronic inflammation.
 12. The compoundaccording to claim 11, wherein the inflammation is induced or caused byinfection, injury, autoimmune disease, or allergy.
 13. The compoundaccording to claim 11, wherein the autoimmune disease is Rheumatoidarthritis.
 14. A method for treatment of inflammation, comprising a stepof administering to a subject in need thereof a therapeuticallyeffective amount of a compound consisting of a conjugate from aglycosaminoglycan and an active compound according to claim
 1. 15. Themethod according to claim 14, wherein the inflammation is induced orcaused by infection, injury, autoimmune disease, or allergy.
 16. Apharmaceutical composition comprising at least one conjugate from aglycosaminoglycan and an active compound according to claim 1 incombination with at least one excipient and/or diluent.
 17. Thepharmaceutical composition according to claim 16, wherein thecomposition is for treating inflammation.
 18. The pharmaceuticalcomposition according to claim 17, wherein the inflammation is inducedor caused by infection, injury, autoimmune disease, or allergy.
 19. Thepharmaceutical composition according to claim 16, wherein thecomposition is designed for oral or injectable administration.